Novel lysophosphatidic acid receptor agonists and antagonists

ABSTRACT

The present invention is directed to compositions comprising lysophosphatidic acid analogs and methods of using such analogs as agonist or antagonists of LPA receptor activity. In addition the invention is directed to LPA receptor agonists that vary in the degree of selectivity at individual LPA receptors (i.e. LPA1, LPA2 and LPA3). More particularly the present invention is directed to LPA analogs wherein the glycerol is replaced with ethanolamine and a variety of substitutions have been linked at the second carbon atom.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 60/516,909 filed on Nov. 3, 2003, thedisclosure of which is incorporated herein in its entirety.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant Nos. NIH R01 GM52722, R01 CA88994, T32 GM07055 and NIH F31 DA05927awarded by National Institutes of Health. The United States Governmenthas certain rights in the invention.

BACKGROUND

Lysophosphatidic acid (LPA, 1-acyl, 2-hydroxyl-sn-glycerol-3-phosphate)is an intermediary metabolite in all cells but is released from somecells to act as a mediator that elicits a wide variety of responses fromcells/tissues. These responses include calcium mobilization,cytoskeletal rearrangements, mitogenesis and anti-apoptotic (survival)activity. For example, LPA is released by activated platelets andaccumulates in serum to low micromolar levels, where it is a prominentgrowth factor for many cell types. LPA has also been found in asciticfluid from ovarian cancer patients where it promotes mitogenesis ofovarian tumor cells. Interestingly, the LPA found in serum vs. asciticfluid differs in that LPA from ascitic fluid is reportedly enriched in2-acyl LPA species. Study of this 2-acyl LPA isoform is made difficulthowever by its chemical instability, i.e., the rapid migration of theacyl chain to the thermodynamically favored 1 position in an aqueousenvironment. Transient rises in blood pressure in rats and guinea pigshas also been documented following intravenous LPA injection. Theinduction of platelet aggregation and fibroblast recruitment along withits mitogenic capabilities implicate this lipid as a wound healinghormone.

LPA signals cells in part via a set of G protein-coupled receptors namedLPA1, LPA2, and LPA3 (formerly Edg-2, Edg-4 and Edg-7). These receptorsshare 50-55% identical amino acids and cluster with five other receptors(S1P1, S1P2, S1P3, S1P4, S1P5 (formerly Edg-1, Edg-5, Edg-3, Edg-6,Edg-8) for the structurally-related lipid sphingosine 1-phosphate (SIP).LPA1 is most associated with activation of Giα pathways and is expressedin oligodendrocytes and peripheral tissues while LPA2 and LPA3 areassociated most prominently with Gq/11α pathways. LPA2 mRNA is found intestis and peripheral blood leukocytes while LPA3 mRNA has beenlocalized to prostate, testes, pancreas, kidney, and heart.

The physiologic implications of occupation of individual LPA receptorsare largely unknown due in part to a lack of receptor-type selectiveligands. Therefore there is a need for compounds that have strongaffinity and high selectivity for LPA receptors. The present inventionis directed to a series of 2-substituted ethanolamide derivatives thatvary in degrees of size, hydrophobicity, and stereochemistry. The parentcompound of the claimed series, N-acyl ethanolamide phosphate (NAEPA)has been shown to be nearly indistinguishable from LPA in evokingplatelet aggregation and GTP[γ₃₅S] binding at LPA1 and LPA2 containingmembranes but is distinctly less active than LPA at recombinant LPA3 orin depolarizing Xenopus oocytes.

Three 2-substituted NAEPA compounds have already been reported. A2-carboxyl-containing compound (named ‘NASPA’ for N-palmitoyl serinephosphate) has been documented to antagonize both LPA-driven plateletaggregation (Sugiura et al., 1994 Arch Biochem Biophys 311: 358-368) andoocyte depolarization (Santos et al., 2000 NYAS Meeting Report; AnnalsNew York Academy of Sciences p 232-241) and is a partial agonist atmammalian LPA receptors. A 2-methylene hydroxy-containing compound,which is an analog of 2-acyl LPA wherein the ester is replaced by anamide, has been reported as activating recombinant LPA receptors in astereoselective fashion while mitogenic responses and plateletaggregation did not show this stereoselectivity. Finally, a thirdcompound (named ‘PNPA’ for N-palmitoyl-norleucinol-1-phosphate) hasn-butyl located at the 2 position. This compound aggregates humanplatelets without regard to stereoselectivity. The present invention isdirected to a series of prodrug derivatives of compounds active at LPAreceptors.

SUMMARY OF VARIOUS EMBODIMENTS OF THE INVENTION

The present invention is directed to a series of LPA analogs wherein theglycerol is replaced with ethanolamine (N-acyl ethanolamide phosphate,N-acyl EPA) as a lead structure. This group of novel compounds, having avariety of substitutions at the second carbon atom of the lead compound,were synthesized and tested for activity at the LPA receptors. LPAanalogs were prepared and found to have a range of activities includingagonism, with various degrees of selectivity at individual LPAreceptors, as well as compounds with antagonist activity at the LPAreceptors. More particularly, one embodiment of the present invention isdirected to phospho-esters of LPA analogs that serve as prodrugs thathave improved oral availability relative to the parent compounds. In oneembodiment the present invention include compounds with the generalstructure:

-   -   wherein R₁ is a large lipophilic group, R₂ and R₃ are various        substituents, n is an integer from 1-10, and R₄ is selected from        the group consisting of hydroxy,    -   wherein R₁₂ is selected from the group consisting of O, NH and        S;    -   X is selected from the group consisting of O, NH, S, CH₂, CHOH,        CHF, CF₂, and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of alkoxy, alkenyloxy, alkynyloxy, aryloxy,        Selective agonists and antagonists at LPA receptors will be        useful therapeutically in a wide variety of human disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the calcium mobilization in A431cells treated with 10 μM of each compound. Columns 1-13 represent theadministration of VPC12086, VPC12101, VPC12109, VPC12115, VPC12098,VPC12105, VPC12084, VPC12255, VPC31144, VPC31143, VPC31180, VPC31139 andLPA, respectively. 100% calcium indicates the signal observed afterpermeabilization with digitonin. Bars are representative of threeexperiments.

FIG. 2A-2D are graphic representations of the effect of VPC12249 andVPC12204 on GTP[γ⁵S] binding. GTP[γ³⁵S] binding assays of LPA1 (FIG.2A), LPA2 (FIG. 2B), and LPA3 (FIG. 2C) transfected HEK293T cellmembranes showing LPA concentration response curves with increasingconcentrations of VPC12249. FIG. 2D shows the effect of VPC12204(enantiomer of VPC 12249) on GTP[γ³⁵S] binding at LP3 transfectedHEK293T cell membranes. Points are in triplicate and are representativeof at least two experiments.

FIG. 3 illustrates dose-response curves for LPA, VPC12031 and VPC12060stimulation of GTPγ[³⁵S] in Rh7777 of HEK293T membranes at LPA1, LPA2,and LPA3 receptors. FIG. 3A represents stimulation of GTPγ[³⁵S] bindingto LPA1 in Rh7777 membranes; FIG. 3B represents stimulation of GTPγ[³⁵S]binding to LPA2 in HEK293T membranes; FIG. 3C represents stimulation ofGTPγ[³⁵S] binding to LPA3 in HEK293T membranes.

FIGS. 4A and 4B illustrate the inhibitory activity of NOHPP (VPC12031)and alpha keto NOHPP analog (VPC12060), respectively, at the LPP1(PAP2a) phosphatase.

FIGS. 5A and 5B illustrate the inhibitory activity of NOHPP (VPC12031)and alpha keto NOHPP analog (VPC12060), respectively, at the LPP2(PAP3a) phosphatase.

FIGS. 6A and 6B illustrate the inhibitory activity of NOHPP (VPC12031)and alpha keto NOHPP analog (VPC12060), respectively, at the LPP3(PAP2b) phosphatase.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect. For example, an effective amount of an S1Preceptor antagonist is an amount that decreases the cell signalingactivity of the S1P receptor.

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term “C₂-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom 2 to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from 2 to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like. The term “C₃-C_(n)cycloalkyl” wherein n=8, represents cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, andthe like. “Optionally substituted aryl” includes aryl compounds havingfrom zero to four substituents, and “substituted aryl” includes arylcompounds having one to three substituents, wherein the substituents,including alkyl, halo or amino substituents. The term (C₅-C₈ alkyl)arylrefers to any aryl group which is attached to the parent moiety via thealkyl group.

The term “heterocyclic group” refers to a mono- or bicyclic carbocyclicring system containing from one to three heteroatoms wherein theheteroatoms are selected from the group consisting of oxygen, sulfur,and nitrogen. As used herein the term “heteroaryl” refers to a mono- orbicyclic carbocyclic ring system having one or two aromatic ringscontaining from one to three heteroatoms and includes, but is notlimited to, furyl, thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7-to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “lower alkyl” as used herein refers to branched or straightchain alkyl groups comprising one to eight carbon atoms, includingmethyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and thelike.

The terms 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers to abranched or straight alkyl or alkenyl group, wherein the first integerrepresents the total number of carbons in the group and the secondinteger represent the number of double bonds in the group.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect. For example, an effective amount of an LPAreceptor antagonist is an amount that decreases the cell signalingactivity of the LPA receptor.

As used herein, an “LPA modulating agent” refers a compound orcomposition that is capable of inducing a detectable change in LPAreceptor activity in vivo or in vitro (e.g., at least 10% increase ordecrease in LPA activity as measured by a given assay such as thebioassay described in Example 2).

As used herein, the term “EC₅₀ of an agent” refers to that concentrationof an agent at which a given activity, including binding of sphingosineor other ligand of an S1P receptor and/or a functional activity of a SIPreceptor (e.g., a signaling activity), is 50% maximal for that SIPreceptor. Stated differently, the EC₅₀ is the concentration of agentthat gives 50% activation, when 100% activation is set at the amount ofactivity of the SIP receptor which does not increase with the additionof more ligand/agonist and 0% is set at the amount of activity in theassay in the absence of added ligand/agonist.

As used herein, the term “phosphate analog” and “phosphonate analog”comprise analogs of phosphate and phosphonate wherein the phosphorousatom is in the +5 oxidation state and one or more of the oxygen atoms isreplaced with a non-oxygen moiety, including for example, the phosphateanalogs phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, boronophosphates, and the like, including associatedcounterions, e.g., H, NH₄, Na, and the like if such counterions arepresent.

The LPA analogs of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the S1P analogs of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the S1P analogs of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstitutedcycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

EMBODIMENTS

Lysophosphatidic acid (LPA) elicits a wide variety of responses fromcells and tissues including calcium mobilization, changes in cell shapeand motility, mitogenesis and anti-apoptosis. These effects are mediatedby at least three LPA receptors (LPA1, LPA2 and LPA3) that have beencloned. Assignment of a physiological response to stimulation of aparticular LPA receptor(s) is made difficult by lack of ligands thatdiscriminate amongst receptor subtypes. The problem is exacerbated bythe existence of at least three lyso-lipid phosphate phosphatases (LPPs)that act as ecto-phosphatases in degrading extra-cellular LPA as well asthe potential for LPA to be acylated by LPA acyl transferases to yieldanother mediator, phosphatidic acid. Therefore, the discovery of newchemical entities that are (1) LPA receptor subtype selective agonistsor antagonist and/or (2) LPA receptor agonists that are resistant toenzymatic degradation and/or (3) inhibitors of the LPPs is highlydesirable.

One embodiment of the present invention is directed to improvedderivatives of LPA analogs, wherein the compounds have been modified toenhance their oral availability and thus increase their efficacy asorally administered pharmaceuticals. The LPA analogs of the presentinvention modulate LPA receptor function through a variety of differentmechanisms including their functioning as a receptor antagonist,receptor agonist (full or partial), or as inhibitors of LPA phosphotasesor synthetic enzymes such as sphingosine kinase or autotoxin. One aspectof the present invention is directed to prodrug derivatives of LPAanalogs, wherein the LPA analogs are prepared as phospho-esterderivatives that have enhanced oral availability relative to the parentcompound. After the compounds are absorbed from the alimentary canal ofthe animal being administered the compound, the phospho-ester is cleavedto regenerate the active form of the compound.

A GTP[735 S] binding assay was developed to analyze directly theactivation of individual LPA receptors, and thus allow theidentification of LPA receptor agonists and antagonists as well asdetermine the relative efficacies and potencies at each receptor in acommon system. The same results were obtained regardless of whether therecombinant receptor used exogenous G proteins (HEK293T cells) orendogenous G proteins (RH7777 cells) and further, the activitiesmeasured in the broken cell assay predicted the responses seen in wholecell assays. Thus the primary assay used in the present invention forcompound potency and efficacy is a valid measure of activity at LPA/Edgreceptors.

Starting with an LPA analog wherein the glycerol is replaced withethanolamine (N-acyl ethanolamide phosphate, N-acyl EPA) as a leadstructure a series of new chemical entities with a variety ofsubstitutions at the second carbon atom were synthesized and tested foractivity at the LPA receptors. In particular, both the relative potencyand efficacy of the 2-substituted N-acyl EPA compounds were measured.The 2-substituted N-acyl EPA compounds of the present invention includecompounds having the general structure:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, C₈-C₂₂ alkanoyl, C₈-C₂₂ alkenoyl,

-   -   wherein m is 0-20;    -   Z is selected from the group consisting of C₃-C₁₀ cycloalkyl,        C₃-C₁₅ bicycloalkyl, C₅-C₁₀ heterocyclic and aryl;    -   R₁₁ is selected from the group consisting of C₁-C₁₀ alkyl,        C₁-C₂₀ alkoxyl, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄        alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄ alkyl)COOR₅,        —(C₁-C₁₀ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic,        C₇-C₁₂ bicyclic, (C₅-C₈ alkyl)aryl, (C₅-C₈ alkenyl)aryl, (C₅-C₈        alkynyl)aryl, and    -   wherein n is 0-10;    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl;    -   R₁₂ is selected from the group consisting of halo, C₁-C₁₀ alkyl,        (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl,        —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆;    -   R₁₃ is selected from the group consisting of H, halo, C₁-C₁₀        alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂        alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆,        NHR₆ and OR₆;        -   wherein R₆ is selected from the group consisting of C₁-C₁₆            alkyl, C₂-C₁₆ alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄ alkyl)R₇,            —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇ and —(C₂-C₄            alkynyl)R₇; and        -   R₇ is selected from the group consisting of optionally            substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈            heterocyclic, optionally substituted C₇-C₁₂ bicyclic and            optionally substituted C₅-C₈ cycloalkenyl and optionally            substituted aryl, wherein the ring structures are            substituted with one or more substituents selected from the            group of C₁-C₄ alkyl, C₁-C₄ alkoxyl, halo, amino or hydroxy            groups;    -   y is 0-4; and        -   R₄ is selected from the group consisting of hydroxy,            phosphonate, and        -   wherein R₁₂ is selected from the group consisting of O, NH            and S;        -   X is selected from the group consisting of O, NH, S, CH₂,            CHOH, CHF, CF₂, and        -   R₃₀ and R₃₁ are independently selected from the group            consisting of C₁-C₂ alkoxy, hydrogen, hydroxy,            and pharmaceutically acceptable salts thereof.

In accordance with one embodiment the LPA analog has the generalstructure

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₉-C₂₂alkenyl,

-   -   wherein m is 0-20;    -   Z is selected from the group consisting of C₃-C₁₀ cycloalkyl,        C₃-C₁₅ bicycloalkyl, C₅-C₁₀ heterocyclic and phenyl;    -   R₁₁ is selected from the group consisting of C₁-C₁₀ alkyl,        C₁-C₂₀ alkoxyl, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄        alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄ alkyl)COOR₅,        —(C₁-C₁₀ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic,        C₇-C₁₂ bicyclic, (C₅-C₈ alkyl)aryl, (C₅-C₈ alkenyl)aryl, (C₅-C₈        alkynyl)aryl, and    -   wherein n is 0-10;    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl;    -   R₁₂ is selected from the group consisting of halo, C₁-C₁₀ alkyl,        (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl,        —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆;    -   R₁₃ is selected from the group consisting of H, halo, C₁-C₁₀        alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂        alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆,        NHR₆ and OR₆;        -   wherein R₆ is selected from the group consisting of C₁-C₁₆            alkyl, C₂-C₁₆ alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄ alkyl)R₇,            —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇ and —(C₂-C₄            alkynyl)R₇; and        -   R₇ is selected from the group consisting of optionally            substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈            heterocyclic, optionally substituted C₇-C₁₂ bicyclic and            optionally substituted aryl, wherein the ring structures are            substituted with one or more substituents selected from the            group of C₁-C₄ alkyl, C₁-C₄ alkoxyl, halo, amino or hydroxy            groups; and    -   R₄ is selected from the group consisting of hydroxy,    -   wherein R₁₂ is selected from the group consisting of O and S;    -   X is selected from the group consisting of O, NH, S, CH₂, CHOH,        CHF, CF₂, and    -    and    -   R₃₀ and R₃, are independently selected from the group consisting        of C₁-C₂ alkoxy, C₂-C₃ alkenyloxy, C₂-C₃ alkynyloxy, aryloxy,    -    and pharmaceutically acceptable salts thereof. In one        embodiment, R₁ is selected from the group consisting of        R₃ is Hand R₄ is    -   wherein X is selected from the group consisting of O, S, CH₂,        CHOH and CHF; and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,        Alternatively, R₁ is selected from the group consisting of

-   R₂ is H and R₄ is    -   wherein X is selected from the group consisting of O, S, CH₂,        CHOH and CHF; and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,    -    In one embodiment a compound of Formula I is provided wherein        R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂        alkenyl, C₈-C₂₂ alkanoyl, C₈-C₂₂ alkenoyl,        -   wherein m is 0-20;        -   Z is selected from the group consisting of C₅-C₆ aryl;        -   R₁₁ is selected from the group consisting of C₁-C₁₀ alkyl;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄        alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, 10-(C₁-C₄ alkyl)COOR₅,        C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic, (C₅-C₈        alkenyl)aryl, (C₅-C₈ alkynyl)aryl and    -    with the proviso that R₂ and R₃ are not both H;        -   wherein n is 0-10;        -   R₅ is selected from the group consisting of H and C₁-C₄            alkyl;        -   R₁₂ is selected from the group consisting of halo, C₁-C₁₀            alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂            alkynyl)aryl, —(C₁-C₄ alkyl)OH, and —(C₂-C₁₂ alkenyl)OH;        -   R₁₃ is selected from the group consisting of H, halo, C₁-C₁₀            alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂            alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆,            SOR₆, NHR₆ and OR₆;    -   y is 0; and    -   R₄ is    -    wherein X is selected from the group consisting of O, S, CH₂,        CHOH and CHF; and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,

In another embodiment of the present invention the LPA analog is acompound represented by the structure:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl, substituted C₈-C₂₂ alkyl and substituted C₈-C₂₂ alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄        alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄ alkyl)COOR₅,        —(C₁-C₁₀ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic,        C₇-C₁₂ bicyclic, (C₅-C₈ alkyl)aryl, (C₅-C₈ alkenyl)aryl, (C₅-C₈        alkynyl)aryl, and    -   wherein n is 0-10;    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl;    -   R₁₂ is selected from the group consisting of halo, C₁-C₁₀ alkyl,        (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl,        —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆;        -   wherein R₆ is selected from the group consisting of C₁-C₁₆            alkyl, C₂-C₁₆ alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄ alkyl)R₇,            —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇ and —(C₂-C₄            alkynyl)R₇; and        -   R₇ is selected from the group consisting of optionally            substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈            heterocyclic, optionally substituted C₇-C₁₂ bicyclic and            optionally substituted C₅-C₈ cycloalkenyl and optionally            substituted aryl, wherein the ring structures are            substituted with one or more substituents selected from the            group of C₁-C₄ alkyl, C₁-C₄ alkoxyl, halo, amino or hydroxy            groups; and    -   R₄ is selected from the group consisting of    -   wherein R₁₂ is selected from the group consisting of O and S;    -   X is selected from the group consisting of O, S, CH₂, CHOH and        CHF; and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,        and pharmaceutically acceptable salts thereof.

In accordance with one embodiment, the LPA analogs of the presentinvention are represented by the structure:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄        alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄ alkyl)COOR₅,        —(C₁-C₁₀ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic,        C₇-C₁₂ bicyclic, (C₅-C₈ alkyl)aryl, (C₅-C₈ alkenyl)aryl, (C₅-C₈        alkynyl)aryl, and    -   R₄ selected from the group consisting of    -   wherein X is selected from the group consisting of O, S, CH₂,        CHOH and CHF; and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl;    -   R₁₂ is selected from the group consisting of halo, C₁-C₆ alkyl,        C₁-C₆ alkyl(C₅-C₈ cycloalkenyl), C₁-C₆ alkenyl(C₅-C₈        cycloalkenyl), (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂        alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkenyl)OH, and OR₆;    -   R₆ is selected from the group consisting of C₁-C₁₆ alkyl, C₂-C₁₆        alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄ carboxy)R₇—(C₁-C₄ alkyl)R₇,        —(C₂-C₄ alkenyl)R₇ and —(C₂-C₄ alkynyl)R₇; and    -   R₇ is selected from the group consisting of optionally        substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈        heterocyclic, optionally substituted C₇-C₁₂ bicyclic and        optionally substituted C₅-C₈ aryl, wherein the ring structures        are substituted with one or more substituents selected from the        group of C₁-C₄ alkyl, C₁-C₄ alkoxyl, halo, amino or hydroxy        groups and pharmaceutically acceptable salts thereof. In one        embodiment, R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂₁ alkenyl, and R₃ is H.        In one embodiment R₁ is a 15:0, 17:0, 17:1, 19:4 or 21:6        hydrocarbon, R₃ is H and R₄ is OPO₃ ⁻² or methylene phosphonate.        The activities of various members of this series have been        tested at the three LPA receptor subtypes and found to have LPA        receptor agonist and antagonist activities.

One embodiment of the present invention is directed to the compounds ofFormula II wherein R₁ is a 15:0, 17:0, 17:1, 19:4 or 21:6 hydrocarbon,R₃ is H, R₄ is OPO₃ ⁻² (or a phosphor-ester derivative thereof) and R₂is selected from the group consisting of 2-substitutions: methyleneamino; para chloro benzyl; methylene benzyl; phenyl; methyl aminobenzyl; aryl, and di-methyl. LPA analogs wherein R₄ is hydroxyl have notdemonstrated activity as LPA receptor agonists or antagonists. However,it is anticipated that such compounds will be phosphorylated in vivoupon administration. Therefore compounds that have activity when R₄ isOPO₃ ⁻² may be formulated as prodrugs by substituting a hydroxy for OPO₃⁻² at R₄. Similarly, the OPO₃ ⁻² group at R₄ can be substituted with aphospho-ester group to form a prodrug that is more readily taken up by apatient's alimentary canal. In addition, the corresponding enantiomersfor all the LPA analogs of the present invention are also encompassed bythe present invention wherein R₁ is a 15:0, 17:0, 17:1, 19:4 or 21:6hydrocarbon, R₄ is selected from the group consisting of

wherein R₃₀ and R₃₁ are independently selected from the group consistingof C₁-C₂ alkoxy,

and R₂ and R₃ are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄alkyl)NH₂, C₀-C₆ alkylhalo, —(C₁-C₁₀ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈heterocyclic, C₇-C₁₂ bicyclic, (C₅-C₈ alkenyl)aryl, (C₅-C₈ alkynyl)aryl,and

-   -   wherein n is 0-3;    -   R₁₂ is selected from the group consisting of H, halo, C₁-C₁₀        alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂        alkynyl)aryl, —(C₁-C₄ alkyl)OH, and —(C₂-C₁₂ alkenyl)OH, with        the proviso that R₂ or R₃ is NH₂, as well as pharmaceutically        acceptable salts thereof. Additional compounds encompassed by        the present invention include:        as well as phospho-ester derivatives of such compounds.

LPA is metabolized by a variety of conceivable routes includingphosphatases, esterases and LPA acyl transferases or transported intocells. The LPA signal at receptors might be prolonged if the routes ofdegradation could be evaded or inhibited by LPA structural analogs. TheLPA analogs of the present invention can be used, in accordance with oneembodiment, to inhibit/evade endogenous LPA metabolic pathways includingphosphotases, esterases, transporters and LPA acyl transferases. Forexample those LPA analogs of Formula I that lack and ester bond would beresistant to degradation by endogenous esterases. One embodiment of thepresent invention is directed to compounds that function as a LPAreceptor agonists and antagonists that are resistant to hydrolysis bylyso-lipid phosphate phosphatases (LPPs) and are sub-type selectiveinhibitors of LPPs. Previously described LPA mimetics contain aphosphate group, and thus are likely susceptible to hydrolysis by LPPs.Furthermore, previously described LPA mimetics have not been shown to beselective for a particular LPA receptor.

Alpha hydroxy phosphonates are well known phosphate mimetics. Forexample, the compounds used clinically to treat osteoporosis(pamidronate, alendronate) are alpha hydroxy bisphosphonates that areanalogs of pyrophosphate. LPA analogs were synthesized wherein thephosphate moiety is replaced by an alpha hydroxy phosphonate. Ratherthan being directly analogous to LPA, the LPP resistant compounds of thepresent invention are analogous to N-oleoyl ethanolamide phosphate—acompound that has been reported to be a potent, efficacious LPA mimetic.The structure of this compound, N-oleoyl-1-hydroxy propylamidephosphonic acid (NOHPP), is as follows:

The IUPAC name of NOHPP is(9Z)-N-(rac-3-hydroxy-3-phosphonopropyl)Octadec-9-enamide. The presentinvention also encompasses phospho-ester prodrug forms of this compoundhaving the structure

wherein R₃₀ and R₃₁ are independently selected from the group consistingof

NOHPP inhibits LPP activity to varying degrees, and is a receptorsubtype selective LPA mimetic. Specifically, NOHPP is fully efficacious(compared to LPA) at the LPA1 and LPA2 receptors, but has littleactivity at the LPA3 receptor. NOHPP is about one log order less potentthan 1-oleoyl LPA at both the LPA1 and LPA2 but greater than two logorders less potent at LPA3. Since the LPA receptor LPA3 has beenreported to be less responsive to LPA with saturated acyl groups (Bandohet al. (1999) J. Biol. Chem. vol. 274, pp. 27776-27785), NOHPP with apalmitoyl group (16:0) might have even greater selectivity for LPA1 andLPA2 vs. LPA3. NOHPP inhibits LPP activities with rank order potencyLPP3>LPP1>>LPP2. Furthermore, the LPA analogs of the present inventionmay function as LPP inhibitors even though they have poor agonist LPAactivity. In accordance with one embodiment the LPP resistant compoundsare used as selective inhibitors of LPP activity.

In addition, further derivatives of NOHPP are encompassed within thepresent invention wherein the amide linkage is replaced with a urealinkage, or an ethane or butane backbone is substituted for the propanebackbone in NOHPP.

In accordance with one embodiment of the present invention a new seriesof compounds has been prepared that are analogous to N-oleoylethanolamide phosphate and are LPP resistant. These LPP resistantcompounds have the general structure:

wherein R¹ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, C₈-C₂₂ alkanoyl, C₈-C₂₂ alkenoyl,

-   -   wherein m is 0-20;    -   Z is selected from the group consisting of C₃-C₁₀ cycloalkyl,        C₃-C₁₅ bicycloalkyl, C₅-C₁₀ heterocyclic and phenyl;    -   R¹¹ is selected from the group consisting of C₁-C₁₀ alkyl,        C₁-C₂₀ alkoxyl, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino;    -   R₂ and R₃ are independently selected from the group consisting        of H, hydroxy, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl,        —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄        alkyl)COOR₅, —(C₁-C₄ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈        heterocyclic, C₇-C₁₂ bicyclic, (C₅-C₁₀ alkyl)aryl, (C₅-C₈        alkenyl)aryl, (C₅-C₈ alkynyl)aryl, and    -   wherein n is 0-10;    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl;    -   R₁₂ is selected from the group consisting of halo, C₁-C₁₀ alky,        (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl,        —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆;    -   R₁₃ is selected from the group consisting of H, halo, C₁-C₁₀        alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂        alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆,        NHR₆ and OR₆;        -   wherein R₆ is selected from the group consisting of C₁-C₁₆            alkyl, C₂-C₁₆ alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄ alkyl)R₇,            —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇ and —(C₂-C₄            alkynyl)R₇; and        -   R₇ is selected from the group consisting of optionally            substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈            heterocyclic, optionally substituted C₇-C₁₂ bicyclic and            optionally substituted C₅-C₈ cycloalkenyl and optionally            substituted aryl, wherein the ring structures are            substituted with one or more substituents selected from the            group of C₁-C₄ alkyl, C₁-C₄ alkoxyl, halo, amino or hydroxy            groups;    -   q is 0-4    -   R₈ and R₉ are independently selected from H, hydroxyl, amino,        COOH, halo, —PO₃; or R₈ and R₉ taken together form a keto group        or a methylene group; and    -   R₁₀ is selected from the group consisting of O, S and NH and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,        In one embodiment, R₁ is C₈-C₂₂ alkanoyl or C₈-C₂₂ alkenoyl, R₂        is H, R₃ is hydroxy, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl,        —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, or —COOR₅ and q is 1.

In one embodiment of the present invention the LPP resistant LPA analogshave the general structure:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, substituted C₈-C₂₂ alkyl and substituted C₈-C₂₂ alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, hydroxy, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl,        —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄        alkyl)COOR₅, —(C₁-C₁₀ alkyl)aryl and        -   wherein n is 1-10;        -   R₅ is selected from the group consisting of H and C₁-C₄            alkyl;        -   R₁₂ is selected from the group consisting of halo, C₁-C₁₀            alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂            alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆,            SORS, NHR₆ and OR₆;            -   wherein R₆ is selected from the group consisting of                C₁-C₁₆ alkyl, C₂-C₁₆ alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄                alkyl)R₇, —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇ and                —(C₂-C₄ alkynyl)R₇; and            -   R₇ is selected from the group consisting of optionally                substituted C₃-C₈ cycloalkyl, optionally substituted                C₃-C₈ heterocyclic, optionally substituted C₇-C₁₂                bicyclic and optionally substituted C₅-C₈ cycloalkenyl                and optionally substituted aryl, wherein the ring                structures are substituted with one or more substituents                selected from the group of C₁-C₄ alkyl, C₁-C₄ alkoxyl,                halo, amino or hydroxy groups; and    -   R₈ and R₉ are independently selected from H, hydroxyl, amino,        COOH, halo, —PO₃; or R₈ and R₉ taken together form a keto group        or a methylene group, and    -   R₃₀ and R₃₁ are independently selected from the group consisting        of C₁-C₂ alkoxy,

In one embodiment of the present invention the LPP resistant LPA analogshave the general structure of Formula IV, wherein R₁ is selected fromthe group consisting of C₈-C₂₂ alkyl, C₈-C₂₂ alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, hydroxy, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl,        —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄        alkyl)COOR₅, —(C₁-C₄ alkyl)aryl and        -   wherein R₅ is H or C₁-C₄ alkyl;        -   R₆ is selected from the group consisting of C₃-C₁₆ alkyl,            C₃-C₁₆ alkenyl, —(C₁-C₄ alkyl)R₇;        -   R₇ is selected from the group consisting of C₃-C₈            cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic and C₅-C₈            aryl; and        -   R₈ and R₉ are independently selected from the group            consisting of H, hydroxyl, amino, COOH, halo, —PO₃; or R₈            and R₉ taken together form a keto group or a methylene group            and R₃₀ and R₃, are independently selected from the group            consisting of C₁-C₂ alkoxy,            In one embodiment, R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂, alkenyl,            and in one embodiment R₁ is a 15:0, 17:0, 17:1, 19:4 or 21:6            hydrocarbon and R₃₀ and R₃, are independently selected from            the group consisting of

In one embodiment, the LPP resistant LPA analog has the generalstructure

-   -   wherein R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂, alkenyl, R₈ and R₉ are        independently selected from the group consisting of H, hydroxyl,        fluoro, or R₈ and R₉ together form a keto group and R₃₀ and R₃₁        are independently selected from the group consisting of C₁-C₂        alkoxy,

In another embodiment the compound has the general structure

wherein R₁ is C₁₄-C₁₈ alkyl or C₁₈-C₂₂ alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, and        -   wherein R₆ is selected from the group consisting of C₃-C₁₆            alkyl, C₃-C₁₆ alkenyl, —(C₁-C₄ alkyl)R₇;        -   R₇ is selected from the group consisting of C₃-C₈            cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic, C₅-C₈            cycloalkenyl and aryl;        -   R₉ is selected from the group consisting of H, hydroxyl,            halo, keto and —PO₃ and R₃₀ and R₃₁ are independently            selected from the group consisting of C₁-C₂ alkoxy,

In accordance with one embodiment compounds of the present inventionhave the general structure:

wherein R₁ is C₈-C₂₂ alkyl or C₈-C₂₂ alkenyl;

-   -   R₂ is selected from the group consisting of C₁-C₆ alkyl, —(C₁-C₄        alkyl)OH, —(C₁-C₄ alkyl)NH₂, COOR₅, —(C₁-C₄ alkyl)COOR₅, and        —(C₁-C₄ alkyl)aryl;    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl.        In one embodiment R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂₁ alkenyl, and R₂        is C₁-C₄ alkyl, methylene hydroxyl, carbomethyl, methylene amino        or benzyl; and    -   R₃₀ and R₃, are independently selected from the group consisting        of C₁-C₂ alkoxy,

In one embodiment compounds that are based on the structure of NOHPP andfall within the scope of the present invention include the followingcompounds:

as well as phospho-ester derivatives of such compounds.

One aspect of the present invention is directed to the NOHPP analog,wls-b8L and phospho-ester derivatives and pharmaceutically acceptablesalts thereof. The IUPAC name for wls-b8L is:(9Z)-N-(3-phosphonopropyl)Octadec-9-enamide, and the trivial name isoleoylaminopropylphosphonate. The structure of wls-b8L is shown below:

wls-b8L is expected to be resistant to hydrolysis by phosphohydrolasesby virtue of its containing a phosphonate, rather than a phosphate,group. Furthermore this chemical entity (wls-b8L) has two additionaldesirable properties. Firstly, wls-b8L is distinctly more selectiveregarding its agonist activity at a single LPA receptor (LPA1) thanVPC122031 (the alpha hydroxy analog of NOHPP) and VPC12060 (the alphaketo analog of NOHPP) and, secondly, at concentrations up to 10micromolar, wls-b8L does not elicit aggregation of human platelets.

The novel LPA receptor agonists disclosed in the present invention areanticipated to have utility in a variety of clinical settings includingbut not limited to the acceleration of wound healing (including cornealwounds), the promotion of myelination (oligodendrocyte cell function)and for immuno-modulation. In particular, LPA has been reported (Balazset al. Am J Physiol Regul Integr Comp Physiol, 2001 280(2):R466-472) ashaving activity in accelerating wound closing and increasingneoepithelial thickness. Accordingly, one embodiment of the presentinvention comprises administering a pharmaceutical compositioncomprising one or more of the LPA receptor agonists of the presentinvention to a mammalian species (including humans) to treat a wound,improve neuronal function or enhance an immune response of that species.LPA has been demonstrated to induce a modest dose-dependent increase inproliferating cells, and a marked increase in the immigration ofhistiocyte-macrophage cells. Accordingly, in one embodiment compositionscomprising an LPA receptor agonist is used to treat wounds, includingburns, cuts, lacerations, surgical incisions, bed sores, andslow-healing ulcers such as those seen in diabetics. In one embodiment acomposition comprising an LPA agonist is administered to a patient toenhance wound repair. Typically the composition is administered locallyas a topical formulation, however other standard routes ofadministration are also acceptable.

The investigation of various 2-substitutions revealed several trends(see Example 2, Table 1) in a series of compounds having the generalstructure of Formula I, wherein R₁ is a 17:1 hydrocarbon and R₄ is OPO₃⁻². First, each LPA receptor showed a marked (one log order or more)preference for one enantiomer. Second, most substitutions were welltolerated in that they resulted in agonist ligands. Third, the LPA3receptor, unlike LPA1 and LPA2, differentiates between unsaturated andsaturated acyl groups and that LPA3 appears to have a lower affinity forLPA and LPA analogs.

Although most active compounds were partial agonists with reducedpotency (relative to LPA), the R-methyl compound (VPC12086) is notablein that it is more potent and efficacious than LPA at LPA1. A patternobserved with all three LPA receptors was that the R configuration wasmore potent with all substituents, an exception is VPC31139, which ismore active in the S configuration. These data indicate that eachreceptor has a single spatial region within the ligand binding site.

In accordance with one embodiment an LPA receptor agonist is providedhaving the general structure

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, substituted C₈-C₂₂ alkyl and substituted C₈-C₂₂ alkenyl;

-   -   R₂ is selected from the group consisting of C₁-C₄ alkyl, C₂-C₄        alkenyl, C₂-C₄ alkynyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂,        —(C₁-C₄ alkyl)COOR₅, and —(C₀-C₁₀ alkyl)aryl;        -   R₄ is represented by the formula    -   wherein R₁₂ is O or S;    -   X is selected from the group consisting of O, NH, S, CH₂, CHOH,        CO₂H, CHF, CF₂, and        -   R₃₀ and R₃₁ are independently selected from the group            consisting of C₁-C₂ alkoxy,        -    and    -   R₅ is selected from the group consisting of H and C₁-C₄ alkyl.

In one embodiment the compound has the general structure:

-   -   wherein R₁ is a saturated or unsaturated, substituted or        non-substituted, straight or branched chain alkyl of about 8 to        22 carbon atoms where the #1 carbon may be in the form of a        carbonyl group (C═O), i.e. the alkyl chain is joined by an amide        linkage. In certain embodiments, R₁ is oleic acid (18:1) or        palmitic acid (16:0) combined in an amide linkage; R₂ is methyl,        ethyl, propyl, isopropyl, butyl, methylene hydroxy, methylene        amino, methylene alkyne, phenyl, benzyl, methylene furan,        methylene-2-naphthalene; and R₄ is hydroxyl (OH), phosphate        (PO₄), or methylene phosphonate (CH₂PO₃). When R₄ is methylene        phosphonate, the carbon alpha to the phosphorus can be        optionally substituted with an hydroxyl, keto, fluoro or        phosphonate moieties or di-substituted with fluoro or hydroxy        and phosphonate substituents, and in further embodiments where        R₄ is a phospho-ester of the general structure    -    wherein R₃₀ and R₃₁ are independently selected from the group        consisting of C₁-C₂ alkoxy,

In accordance with one embodiment the LPA receptor agonist is providedhaving the general structure:

-   -   wherein R₂ is methyl, ethyl, propyl, isopropyl, butyl, methylene        hydroxy, methylene amino, methylene alkyne, phenyl, benzyl,        methylene furan, methylene-2-naphthalene; and R₄ is a        phospho-ester of the general structure        wherein R₃₀ and R₃₁ are independently selected from the group        consisting of C₁-C₂ alkoxy,

In accordance with one embodiment of the present invention LPA receptorsubtype-selective compounds having agonist and/or antagonist propertiescan be administered to a subject to treat or prevent a disorder ofabnormal cell growth and differentiation. These disorders include, butare not limited to, Alzheimer's disease, aberrant corpus luteumformation, osteoarthritis, osteoporosis, anovulation, Parkinson'sdisease, multiple sclerosis and rheumatoid arthritis.

As noted above LPA3 has a lower affinity for LPA and LPA analogs. It isbelieved that LPA3 may play a role in feedback inhibition of activity atLPA1 and LPA2. Therefore it is anticipated that an LPA3subtype-selective agonist can be used to decrease LPA1 and LPA2 mediatedactivities. Thus in accordance with one embodiment an LPA3subtype-selective agonist can be administered to a subject to treat orprevent a disorder of abnormal cell growth and differentiation,including cancer. To be considered a subtype selectiveagonist/antagonist, the compound should be 10× more potent, and morepreferably 100× more potent, at the preferred LPA receptor. For example,it appears that mono-unsaturated substituents at R₁ of the LPA receptoranalogs of Formula I are significantly more potent (equipotent to LPA)than the saturated compound at LPA3. LPA1 and LPA2 do not exhibit thisselectivity for unsaturated vs. saturated side chains.

Similarly LPA receptor antagonist can also be used to inhibit LPAmediated activities and thus treat disorders of abnormal cell growth anddifferentiation as well as inflammatory diseases. These disordersinclude, but are not limited to, Alzheimer's disease, aberrant corpusluteum formation, osteoarthritis, osteoporosis, anovulation, Parkinson'sdisease, multiple sclerosis, rheumatoid arthritis and treatment ofcancer. Such antagonists can then be formulated as pharmaceuticalcompositions using standard pharmaceutically acceptable carriers,fillers, solublizing agents and stabilizers known to those skilled inthe art. Pharmaceutical compositions comprising the LPA receptorsubtype-selective agonist and/or antagonist are administered to anindividual in need thereof by any number of routes including, but notlimited to, topical, oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

In accordance with one embodiment LPA receptor antagonists are provided,wherein the antagonists have the general structure

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, C₈-C₂₂ alkanoyl, C₈-C₂₂ alkenoyl,

-   -   wherein m is 0-20;    -   Z is selected from the group consisting of C₃-C₁₀ cycloalkyl,        C₃-C₁₅ bicycloalkyl, C₅-C₁₀ heterocyclic and phenyl;    -   R₁₁ is selected from the group consisting of C₁-C₁₀ alkyl,        C₁-C₂₀ alkoxyl, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino;    -   R₂ and R₃ are independently selected from the group consisting        of H, and        -   wherein n is 0-10;        -   R₁₂ is selected from the group consisting of C₁-C₁₀ alkyl,            (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂            alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆,            SOR₆, NHR₆ and OR₆;        -   R₁₃ is selected from the group consisting of H, C₁-C₁₀            alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂            alkynyl)aryl, —(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH,            phenyl-4-methoxy, SR₆, SOR₆, NHR₆ and OR₆;            -   wherein R₆ is selected from the group consisting of                C₁-C₁₆ alkyl, C₂-C₁₆ alkenyl, C₂-C₁₆ alkynyl, —(C₁-C₄                alkyl)R₇, —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇ and                —(C₂-C₄ alkynyl)R₇;            -   R₇ is selected from the group consisting of optionally                substituted C₃-C₈ cycloalkyl, optionally substituted                C₃-C₈ heterocyclic, optionally substituted C₇-C₁₂                bicyclic, optionally substituted C₅-C₈ cycloalkenyl and                optionally substituted aryl; and        -   R₄ is        -   wherein R₁₂ is selected from the group consisting of O and            S;        -   and R₃₀ and R₃₁ are independently selected from the group            consisting of            In one embodiment n is 1, R₁₃ is H and R₁₂ is SR₆, SOR₆,            NHR₆ or OR₆.

In accordance with one embodiment LPA receptor antagonists are provided,wherein the antagonists have the general structure

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₉-C₂₂alkenyl, substituted C₈-C₂₂ alkyl and substituted C₈-C₂₂ alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, and        -   wherein R₆ is selected from the group consisting of C₃-C₁₆            alkyl, C₃-C₁₆ alkenyl, —(C₁-C₄ alkenyl)R₇, —(C₂-C₄            alkynyl)R₇ and —(C₂-C₄ alkyl)R₇;    -   R₇ is        -   wherein R₁₄ and R¹⁵ are independently selected from the            group consisting of H, C₁-C₁₂ alkyl, C₂-C₆ alkenyl, C₂-C₆            alkynyl, halo; and        -   R₄ is        -   wherein R₁₂ is selected from the group consisting of O and            S;        -   and R₃₀ and R₃₁ are independently selected from the group            consisting of            with the proviso that R₂ and R₃ are not both H. In one            embodiment, R₁ is C₁₃-C₁₇ alkyl or

-   C₁₇-C₂₁ alkenyl and R₄ is    -   wherein R₃₀ and R₃₁ are independently selected from the group        consisting of

In one embodiment R¹, is a 15:0, 17:0, 17:1, 19:4 or 21:6 hydrocarbonand R₄ is

-   -   wherein R₃₀ and R₃₁ are independently selected from the group        consisting of

In accordance with one embodiment the LPA receptor antagonist has thegeneral structure:

-   -   wherein R₂ and R₃ are independently selected from the group        consisting of H, benzyl, methylene furan,        methylene-2-naphthalene, methylene phenyl-4-O-benzyl, methylene        phenyl-4-benzyl, methylene phenyl-4-chloro, methylene        phenyl-4-trans-styrene, methylene phenyl-4-cis-styrene,        methylene phenyl-4-O-2,6-dichlorobenzyl and methylene        phenyl-4-phenyl. In one embodiment R₂ is H and the compound is        prepared as a phospho-ester derivatives thereof.

Additional compounds that serve as antagonists of LPA receptor functioninclude compounds of the general formula:

-   -   wherein R₃₀ and R₃₁ are independently selected from the group        consisting of C₁-C₂ alkoxy,    -    and R₃ is selected from the group:

Compound Name and Stereochemistry Compound name and Stereochemistry (R)(S)

VPC12204 VPC12249

VPC12250 VPC12229

VPC12193 VPC12227

VPC12235 VPC12228

VPC12284

VPC13061 VPC13082

VPC13063 VPC13086

VPC13066

VPC13069 VPC13089

One example of an LPA antagonist in accordance with the presentinvention is VPC12249. That compound, having the backbone structure ofFormula II, wherein R₁ is a 17:1 hydrocarbon, R₂ is H, R₄ is OPO₃ ⁻² (ora phosphos-ester or pharmaceutically acceptable salt thereof) and R₃contains the benzyl-4-oxybenzyl functionality in the S-configuration,which confers antagonistic activity at LPA1 and LPA3 receptors.VPC12249, to our knowledge, is the first specific LPA1/LPA3 receptorantagonist. This compound possesses high affinity for the LPA1 (ca.K_(I)=130 nM) and LPA3 (ca. K_(I)=425 nM) receptors. In additioncompound VPC31162 (structure of Formula II, wherein R₁ is a 17:1hydrocarbon, R₂ is H, R₄ is OPO₃ ⁻² (or a phosphos-ester orpharmaceutically acceptable salt thereof) and R₃ isbenzyl-4-oxybenzyl-4-pentane) has antagonist activity at the LPA2receptor, but is an agonist at LPA1 and LPA3.

In accordance with one embodiment of the present invention the LPAanalogs that exhibit LPA receptor antagonist activity can be used totreat ischemia reperfusion type injury. Interference with the supply ofoxygenated blood to tissues is defined as ischemia. The effects ofischemia are known to be progressive, such that over time cellularvitality continues to deteriorate and tissues become necrotic. Totalpersistent ischemia, with limited oxygen perfusion of tissues, resultsin cell death and eventually in coagulation-induced necrosis despitereperfusion with arterial blood. A substantial body of evidenceindicates that a significant proportion of the injury associated withischemia is a consequence of the events associated with reperfusion ofischemic tissues, hence the term reperfusion injury.

To place reperfusion injury into a clinical perspective, there are threedifferent degrees of cell injury, depending on the duration of ischemia:(1) With short periods of ischemia, reperfusion (and resupply of oxygen)completely restores the structural and functional integrity of the cell.Whatever degree of injury the cells have incurred can be completelyreversed upon reoxygenation. (2) With longer periods of ischemia,reperfusion is not associated with the restoration of cell structure andfunction, but rather with deterioration and death of cells. The responseto reoxygenation in this case is rapid and intense inflammation. (3)Lethal cell injury may develop during prolonged periods of ischemia,where reperfusion is not a factor. The reversibility of cell injury as aconsequence of ischemia is determined not only by the type and durationof the injury, but also by the cell target. Neurons exhibit very highsensitivity to ischemia, whereas myocardial, pulmonary, hepatic andrenal tissues are intermediate in sensitivity. Fibroblasts, epidermisand skeletal muscle have the lowest susceptibility to ischemic injury,requiring several hours without blood supply to develop irreversibledamage.

In accordance with the present invention a compound having the generalstructure:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, substituted C₈-C₂₂ alkyl and substituted C₈-C₂₂ alkenyl;

-   -   R₂ and R₃ are independently selected from the group consisting        of H, and        -   wherein R₆ is selected from the group consisting of C₃-C₁₆            alkyl, C₃-C₁₆ alkenyl, —(C₁-C₄ alkenyl)R₇, —(C₂-C₄            alkynyl)R₇ and —(C₂-C₄ alkyl)R₇;        -   R₇ is        -   wherein R₁₄ and R¹⁵ are independently selected from the            group consisting of H, C₁-C₁₂ alkyl, C₂-C₆ alkenyl, C₂-C₆            alkynyl, halo; and        -   R₄ is        -   wherein R₁₂ is selected from the group consisting of O and            S;        -   and R₃₀ and R₃₁ are independently selected from the group            consisting of            with the proviso that R₂ and R₃ are not both H is            administered to reduce of prevent reperfusion injury. In one            embodiment, R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂, alkenyl and R₄ is    -   wherein R₃₀ and R₃₁ are independently selected from the group        consisting of

In one embodiment R₁ is a 15:0, 17:0, 17:1, 19:4 or 21:6 hydrocarbon andR₄ is

-   -   wherein R₃₀ and R₃₁ are independently selected from the group        consisting of        In one embodiment the compound is VPC12249.

The LPA receptor antagonist of the present invention can be formulatedwith pharmaceutically acceptable carriers, diluents, and solubilizingagents for administration to patient in need of such therapy. Thecompounds are preferably administered intravenously, but any standardroute of administration is acceptable including oral delivery. Inparticular, if the LPA receptor antagonist is administered prior totissue injury, such as to a patient prior to surgery, the LPA receptorantagonist can be administered orally.

In one embodiment a therapeutically effective amount of the LPA receptorantagonist is administered intravenously in a physiologically acceptablecarrier as early as possible, and most preferably within four hours of areperfusion injury. Subsequent doses of the LPA receptor antagonist, canbe administered intravenously or orally.

EXAMPLE 1

Synthesis of 2-Substituted, Ethanolamide-Based LPA Analogs

Chemicals for syntheses were purchased from Aldrich Chemical Company,Inc., Sigma Chemical Company, Inc., Advanced ChemTech Chemical Company,Inc. and/or NovaBiochem Chemical Company, Inc., and were used withoutfurther purification. The general approach used to synthesize the LPAanalogs of the present invention is shown in Scheme 1. Briefly the sevensteps are as follows:

General Procedure A: Installation of Chiral Auxillary

To a solution of carbobenzyloxy-protected amino acid in diethyl ether at−78° C. is added triethylamine followed by pivaloyl chloride. Theresulting thick white precipitate is stirred for 1 hr at 0° C. and thenre-cooled to −78° C. In a separate flask, a solution of[4S]-4-benzyl-2-oxaxolidinone in tetrahydrofuran (THF) is prepared. Oncooling this solution to −78° C., n-butyllithium is added via a syringeover 5 minutes. The resulting solution is cannulated to the flaskcontaining the mixed anhydride. The mixture is stirred for 15 min at−78° C. and 30 min at 0° C. before quenching by addition of aqueousammonium chloride. The resulting solution is concentrated and dilutedwith methylene chloride, extracted (3×) with sodium bicarbonate andbrine and dried (Na₂SO₄). The imide product is isolated by flashchromatography.

General Procedure B: Alpha-Carbon Alkylation

To a solution of imide in dichloromethane at 0° C. is added titaniumtetrachloride. After stirring for 5 minutes, diisopropylethylamine isadded. The solution is stirred for 3 hours at 0° C., and then thealkylbromide is added. The resulting solution is stirred for anadditional 2 hours before cannulated to a vigorously stirring mixture ofsaturated aqueous sodium bicarbonate. The layers are separated andextracted (three times) with dichloromethane, dried (Na₂SO₄) andconcentrated. The α,α-substituted product is isolated by flashchromatography.

General Procedure C: Chiral Auxilliary Removal

To a solution of α,α-substituted amino acid in THF at 0° C. is addedmethanol and lithium borohydride. The solution is stirred for 1 hour andquenched by the addition of 1.0M sodium potassium tartrate and stirringfor 10 min at 0° C. The layers are separated and extracted (3×) withdichloromethane, dried (Na₂SO₄) and concentrated. The alcohol product isisolated by flash chromatography.

General Procedure D: Phosphorylation

To a solution of alcohol in 1:1 THF/CH₂Cl₂ in an aluminum foil coveredround bottom flask is added tetrazole. After stirring for 15 minutes,di-t-butyldiisoproylphosphoramidite is added. The solution is stirredfor 6 hours and quenched with sodium metabisulfite at 0° C., andextracted with ethylacetate. The organic layers are combined, dried(Na₂SO₄) and concentrated. The product is purified by flashchromatography.

General Procedure E: N-Cbz Deprotection

To a solution of Cbz-protected phosphate in ethanol is added 10% Pd/C.The resulting solution is placed under H₂ atmosphere. After 2 hours, thesolution is filtered and concentrated to provide the product.

General Procedure F: Addition of Fatty Acid Moiety

To a solution of amine in dichloromethane is addedN,N-diisopropylethylamine. After stirring for 10 minutes, oleoylchloride is added via a syringe over 20 minutes. The resulting solutionis quenched by adding saturated aqueous ammonium chloride after 1 hour.The aqueous layer is extracted (3×) with ethyl acetate and the combinedorganic layers are dried (Na₂SO₄) and concentrated. The product ispurified by flash chromatography.

General Procedure G: Phosphate Deprotection

To a solution of protected phosphate in dichloromethane is addedtrifluoroacetic acid. After 2 hours, the solvent is removed via rotaryevaporation and ether is added and removed. This process is repeateduntil the all trifluoroacetic acid is removed, providing the phosphatedeprotected product.

Additional synthetic details are provided in-the following:

Reagents: (a) i. DIEA, isobutylchloroformate, ii. NaBH₄, H₂O; (b)1H-Tetrazole, N,N-di-t-butyl diisopropylphosphoramidite; (c) 10% Pd/C,H₂; (d) Oleoyl chloride, DIEA or pyridine; (e) 1:1 TFA/CH₂Cl₂

Reagents: (a) Oleoyl chloride, DIEA or pyridine; (b) 1H-Tetrazole,N,N-di-t-butyl diisopropylphosphoramidite; (c) 1:1 TFA/CH₂Cl₂

Reagents: (a) i. DIEA, isobutylchloroformate, ii. NaBH₄, H₂O; (b) 1:1TFA/CH₂Cl₂; ii. Oleoyl chloride, DIEA or pyridine; (c) 1H-Tetrazole,N,N-di-t-butyl diisopropylphosphoramidite; (d) 10% Pd/C, H₂; (e) 1:1TFA/CH₂Cl₂

Reagents: (a) i. DIEA, isobutylchloroformate, ii. NaBH₄, H₂O; (b)1H-Tetrazole, N,N-di-t-butyl diisopropylphosphoramidite; (b) i. 1:1DIEA/CH₂Cl₂; ii. Oleoyl chloride; (d) 1:1 TFA/CH₂Cl₂

Reagents: (a) BnNH₂, NaBH₃CN, MeOH; (b) 15% Pd(OH)₂, H₂; (c) i. Fmoc-Cl,NaHCO₃, THF/H₂O; ii. 1:1 TFA/CH₂Cl₂, then HCl; (d) Oleoyl chloride,DIEA; (e) 1H-Tetrazole, N,N-di-t-butyl diisopropylphosphoramidite; (f)i. piperidine, DMF; ii. 1:1 TFA/CH₂Cl₂

EXAMPLE 2

Activity of 2-Substituted LPA Analogs at Edg/LPA Receptors

Materials and Methods

Transient Expression in HEK293T Cells

The appropriate receptor plasmid DNA (encoding mouse LPA1, human LPA2 orhuman LPA3) was mixed with equal amounts of expression plasmids (pcDNA3)encoding a mutated (C351F) rat Gi2a, cow β1, and γ2 proteins and theseDNAs were used to transfect monolayers of HEK293T cells (where ‘T’indicates expression of the SV-40 virus large T antigen) using thecalcium phosphate precipitate method. After about 60 hours, cells wereharvested, membranes were prepared, aliquoted and stored at −70° C.until use.

GTP[35 γS] Binding:

The GTP[35 γS] assay was performed as described by us previously.Membranes containing 5 ug of protein were incubated in 0.1 mlGTP-binding buffer (in mM: HEPES 50, NaCl 100, MgCl₂ 10, pH7.5)containing 5 mg saponin, 10 mM GDP, 0.1 nM GTP[35 γS] (1200 Ci/mmol),and indicated lipid(s) for 30 minutes at 30° C. Samples were analyzedfor membrane-bound radionuclide using a Brandel Cell Harvester(Gaithersburg, Md.). The C351F mutation renders the Gi2a proteinresistant to inactivation by pertussis toxin or the alkylating agentN-ethylmaleimide; however in practice background binding wassufficiently low to obviate these maneuvers.

Measurement of cAMP Accumulation:

Assays for cAMP accumulation were conducted on populations of 5×10⁵cells stimulated with 10 mM forskolin in the presence of thephosphodiesterase inhibitor isomethylbutylxanthine (IBMX) for 15 minutesat 30° C. cAMP was measured by automated radioimmunoassay.

Measurement of Intracellular Calcium:

A FLIPR™ (Molecular Devices, Inc.) was used to measure intracellularcalcium in A431 and HEK293T cells. A431 cells were seeded (−50,000cells/well) in 96-well clear bottom black microplates (Corning CostarCorp., Cambridge, Mass.) and left overnight in CO₂ incubator at 37° C.HEK293T cells were treated likewise, but seeded onto poly D-lysinecoated microplates (Becton Dickinson, Franklin Lakes, N.J.). A431 cellswere dye-loaded with 4 μM Fluo-3 AM ester (Molecular Probes Inc.,Eugene, Oreg.) in a loading buffer (1×HBSS buffer, pH 7.4, containing 20mM HEPES, 0.1% BSA, and 2.5 mM probenecid) for 1 hour at 37° C. Cellswere then washed four times with the loading buffer and exposed in theFLIPR™ to sets of compounds. HEK293T cells were loaded with 2 μM Fluo-4AM ester (Molecular Probes Inc., Eugene, Oreg.) in the same loadingbuffer without probenecid for 30 minutes and washed four times beforebeing exposed to compounds in the FLIPR™. In all cases, eachconcentration of each compound was tested in at least quintuplicate.

Determination of κ1:

κ1 for VPC12249 in experiments were determined by plotting the log ofDose Ratio-1 at each concentration of inhibitor against the logconcentration of inhibitor. The X-intercept of the linear transformationis equal to the inverse log of the κ1.

Stable Expression in RH7777 Cells:

Rat hepatoma RH7777 cell monolayers were transfected with the mLP_(A1)plasmid DNA using the calcium phosphate precipitate method and clonalpopulations expressing the neomycin phosphotransferase gene wereselected by addition of geneticin (G418) to the culture media. TheRH7777 cells were grown in monolayers at 37° C. in a 5% CO02/95% airatmosphere in growth media consisting of: 90% MEM, 10% fetal bovineserum, 2 mM glutamine and 1 mM sodium pyruvate.

Cardiovascular Measurements:

All procedures were performed on Male Wistar rats in accordance withNational Institutes of Health and University of Virginia animal care andusage guidelines. Anesthesia was induced by 5% halothane (in 100% O₂).Rats were intubated and artificially ventilated with 1.5-1.8% halothanein 100% O₂ for surgical procedures. A femoral artery was cannulated torecord mean arterial pressure (MAP) and heart rate (HR), and a femoralvein was cannulated to administer anesthetic agents. A femoral vein wascannulated for administration of lipids. The left splanchnic nerve wasisolated via a retroperitoneal approach, and the segment distal to thesuprarenal ganglion was placed on two Teflon-coated silver wires thathad been bared at the tip (250 mm bare diameter; A-M Systems, Everett,Wash.). The nerve and wires were embedded in a dental impressionmaterial (polyvinysiloxane; Darby Dental Supply, Westbury, N.Y.), andthe wound was closed around the exiting recording wires. On completionof surgery, the halothane anesthesia was terminated and was replaced bya α-chloralose (30 mg/kg solution in 3% sodium borate; 70 mg/kg initialbolus followed by hourly supplements of 20 mg/kg iv; Fisher Scientific,Pittsburgh, Pa.). Rats were allowed to stabilize for 45 min before testsbegan. End-tidal CO₂ was monitored by infrared spectroscopy and wasmaintained between 3.5 and 4.0%. Body temperature (measured rectally)was maintained at 37° C.

All physiological variables were monitored on a chart recorder (model RS3600, Gould, Valley View, Ohio) and simultaneously stored on avideocassette recorder via a digitizer interface (model 3000A, frequencyrange: DC-22 kHz; Vetter Digital, Rebersburg, Va.) for off-line computeranalysis. Data was analyzed with Spike 2 (Cambridge Electronics). TheMAP was calculated from the pulse pressure measured by a transducer(Statham P10 EZ, Gould) connected to the brachial arterial catheter. TheHR was determined by triggering from the pulse pressure (Biotach,Gould). Splanchnic nerve activity (SNA) was filtered (10 Hz-3 kHz bandpass with a 60-Hz notch filter), full-wave rectified, and averaged in1-s bins. The femoral venous catheter (dead space 1100 mL) was loadedwith each lipid and was flushed with 200 mL of saline to expel the drug.

Materials: Chemicals for syntheses were purchased from Aldrich ChemicalCompany, Inc., Sigma Chemical Company, Inc., Advanced ChemTech ChemicalCompany, Inc. and/or NovaBiochem Chemical Company, Inc., and were usedwithout further purification. GTP[γ³⁵S] was purchased from Amerhsam,Fura-3 and Fura-4 AM were purchased from Molecular Probes Inc., A431 andRH7777 cells were purchased from the American Type Culture Collection(Manassas, Va.) and tissue culture media and serum was fromGibcoBRL/Life Technologies (Gaithersburg, Md.). HEK293T cells were agift from Dr. Judy White's laboratory (Dept. Cell Biology, University ofVirginia) while G protein P and y DNAs were a gift from Dr. Doug Bayliss(Dept. Pharmacology, University of Virginia). LPAs (1-oleoyl and1-palmitoyl) and dioctyl glyceryl pyrophosphate were purchased fromAvanti Polar Lipids (Alabaster, Ala.).

Results:

Using N-oleoyl ethanolamide phosphoric acid (NAEPA) as a lead structure,a series of 2-substituted LPA analogs was synthesized. The details ofthe synthesis and analysis of the full set of compounds in this seriesis described in Example 2. Each compound was characterized by ¹H NMR,¹³C NMR, and mass spectrometry.

The differential coupling of LPA1 versus LPA2 and LPA3, the lack of areliable radioligand binding assay, and the near ubiquity of endogenousLPA responses prohibited the use of most common receptor assaytechniques (i.e. measurements of adenylyl cyclase activity, calciummobilization, radioligand binding) to assess each compound's activity.Therefore, we adapted a GTP[γ³⁵S] binding assay to measure the relativeefficacies and potencies of each compound compared to LPA. This assayisolates each recombinant LPA receptor and allows analysis of all threereceptors using the same system. Note that membranes from HEK293T cellstransfected with only G protein DNAs (i.e., no receptor DNA) were devoidof LPA-stimulated GTP binding despite expressing endogenous LPAreceptors.

Many NAEPA compounds with various 2-substituents were synthesized andexamined. Since the 2 position is a prochiral site, both enantiomers ofthe eight compounds were synthesized. Three patterns were revealed whenthe agonist compounds were tested in this series at the three LPAreceptors in the broken cell assay. First, each LPA receptor showed amarked (one log order or more) selectivity for one enantiomer. Second,those compounds with substitutions at the R₂ position of Formula I wereinvariably the more potent agonists. Third, agonist potency decreases asthe bulk of the substituent increases. The 2-substituted NAEPA compoundscontaining either hydrophilic (methylene hydroxy, carbomethyl, methyleneamino) or hydrophobic moieties (methyl, ethyl, isopropyl, benzyl)exhibited agonist activity in the GTP[γ³⁵S] binding assay (see Table 1).The smaller groups conferred greater potency, with the methyl(VPC12086), methylene hydroxy (VPC31143) and methylene amino (VPC12178)compounds being more potent than 1-oleoyl LPA at LPA1 (Table 1). Also,as the 2-substituent becomes bulkier, the efficacy was noticeablyreduced at this receptor. In contrast, bulkier hydrophobic side chains,although less potent, were fully efficacious at the LPA2 receptor (Table1). TABLE 1 Agonist activities of 2-substituted N-oleoyl EPA compoundsA1 A2 A3 LP LP LP Functional EC50 EC50 EC50 Lipid Group (nM) Emax (nM)Emax (nM) Emax LPA 11.7 100 6.8 100 262.5 100 31143 Methylene 7.9 161.4116.5 104.6 321.8 106.6 Hydroxy 31144 Methylene >5000 91.1 2645 77.34349 N/A Hydroxy 31139 Carbo 18.5 66.2 29.2 91.3 1484 84.4 Methyl 31180Carbo 1215 50.6 3461 60.2 >5000 3.8 Methyl 12178 Methylene 4.9 98.7 50.3103.2 683.7 102.9 Amino 12048 Methylene >5000 9.4 >5000 36.4 >5000 3.8Amino 12086 Methyl 3.4 107.6 18.3 95.2 112.6 93.4 12101 ″ 290048.1 >5000 6.9 >5000 9.7 12109 Ethyl 35.8 66.8 161.9 92.6 1083 83.512115 ″ 1580 62.3 4280 85.6 4980 89.3 12098 Isopropyl 73.8 51 799.3 85.83815 45.3 40105 ″ >5000 14.8 >5000 27.6 >5000 27.7 12084 Benzyl 38.423.7 268.3 97 351.4 78.7 12255 ″ >5000 N/A >5000 N/A >5000 N/A

As was observed with the LPA1 receptor, the small methyl and methyleneamino groups conferred the highest potency at the LPA2 receptor but noneof these compounds proved more potent than 1-oleoyl LPA at this site.The LPA3 receptor exhibited much the same profile as the LPA2 receptoras far as efficacies and potencies of compounds relative to LPA areconcerned. However, the LPA3 receptor characteristically exhibitedhigher (1-2 log order) EC₅₀ values for all compounds, including LPA.Presumably, the LPA3 receptor has an intrinsically lower affinity forLPA and LPA analogs. Like the hydrophilic compounds, each LPA receptorshowed strong stereoselectivity for a hydrophobic substituent in the R₂position of Formula I.

Although saturated ligands were repeatedly found that are active atLPA3, mono-unsaturated compounds were also consistently found that aremore potent at this receptor. For example, preliminary results with an18:1 (oleoyl) analog of SDB-213 suggest that the mono-unsaturatedcompound is significantly more potent (equipotent to LPA) than thesaturated compound at LPA3 in this assay. LPA1 and LPA2 do not exhibitthis selectivity for mono-unsaturated vs. saturated acyl groups.

To investigate an LPA response in a physiologic context, mean arterialblood pressure (MAP), heart rate, and postganglionic sympathetic tonewas monitored in anesthetized adult rats as a function of LPA or LPAanalog administration. LPA had been shown previously to increase bloodpressure transiently in this model. Intravenous injection of threeenantiomeric pairs of compounds resulted in a transient increase in MAPwith the same pattern of stereoselectivity as observed with the in vitroassays. Concomitant with this rise in MAP was a decrease in heart rateand sympathetic output indicative of baroreceptor reflex response.

The compounds that were only slightly efficacious at LPA1, e.g. thebenzyl-containing VPC12084, were assayed for their ability to antagonizeLPA induced GTP[γ₃₅ S] binding. Although this compound did block LPAactivity in the GTP[γ³⁵ S] binding assay, the benzyl compound (VPC12084)was revealed to posses appreciable agonist activity in assays withgreater levels of amplification (e.g., whole cell assays of calciummobilization or inhibition of cAMP accumulation). In the course ofexploring variations of the benzyl substituent, a benzyl-4-oxybenzylsubstituent in the same relative configuration (R₂: VPC12204) was foundto have a reduced, but still measurable, agonist activity in whole cellassays. However, its enantiomer [i.e., VPC12249, the compound with thebenzyl-4-oxybenzyl substituent in the S (the R₃ substituent of FormulaI) configuration] was completely devoid of agonist activity in the wholecell assays and in the GTP[γ₃₅ S] binding assay. VPC12249 was tested forits ability to block LPA-induced GTP[γ₃₅ S] binding at each recombinantLPA receptor. As is shown by the rightward, parallel shifts in theconcentration response curves as a function of VPC12249 concentration,this compound is a surmountable antagonist at the LPA1 and LPA3, but notthe LPA2, receptors (FIG. 6). The κ1 values for VPC12249 determined bySchild regression are 137 and 428 nM at the LPA1 and LPA3 receptors,respectively, in this assay. The same activity was determined with humanLPA1 using a recombinant baculovirus-infected insect Sf9 cell membranepreparation.

The antagonist activity measured in the broken cell assays was confirmedin whole cell experiments wherein LPA-induced rises in freeintracellular calcium in HEK293T cells were blocked. This cell typeexpresses the LPA1 and LPA3, but not LPA2 receptor genes, as determinedby RT-PCR. Increasing concentrations of VPC12249 resulted in parallel,rightward shifts in the LPA concentration response curves (κ1 132 nM).The extent of rightward shift observed in the same experimental protocolwith A431 cells, which express the LPA2 as well as the LPA1 and LPA3genes (RT-PCR not shown), was much smaller (κ1 1970 nM) as predictedfrom the lack of antagonist activity of VPC12249 at thecalcium-mobilizing LPA2 receptor in the GTP binding assay. The blockingaction of VPC12249 was not a general post-receptor event as shown by thelack of antagonism of ATP-evoked calcium transients. Inhibition offorskolin-induced increases in cAMP levels in RH7777 cells stablyexpressing LPA1 was also inhibited by VPC12249.

EXAMPLE 3 Synthesis of rac-N-oleoyl-1-hydroxy-propylamide PhosphonicAcid (NOHPP)

Two batches of NOHPP have been synthesized as a racemic mixture. Afterobserving LPA mimetic activity with the first batch (named JAR1842), thematerial was re-synthesized, and this batch was named VPC12031. Bothbatches showed the same profile on thin layer chromatography (TLC)(R_(f) 0.31, chloroform/acetone/methanol/glacial acetic acid/water,50/15/13/12/4) whereon they migrated as a single, discrete spot.Further, the NMR spectrum of VPC12031 was as expected and the formulaweight measured by mass spectrometry (419.5 daltons) agrees with that ofthe structure. Thus confirming the identity and high degree of purity ofthe NOHPP sample.

To assay NOHPP and other compounds at individual LPA receptors, theadapted GTPγS binding assay of Example 3 was used. Briefly, individual,recombinant LPA receptors were express along with heterotrimeric Gproteins in HEK293T cells. For the LPA receptor LPA 1, which naturallycouples to Gi/o proteins, membranes from rat hepatoma cells (RH7777)were also used that have been transfected only with LPA1 DNA. Membranesprepared from these cells are used to measure GTPγ[³⁵S] binding as afunction of test compound concentration. The resulting dose-responsecurves provide relative potency (EC₅₀) and efficacy, (E_(max)) for thetest compounds. The assay is robust and free of background from the LPAreceptors endogenous to HEK293T cells. In FIGS. 2A-C we show therelative activities of LPA and NOHPP at the three known (i.e. cloned)LPA receptors (LPA1, LPA2 and LPA3). NOHPP is distinctly less potent andfar less efficacious than LPA at LPA3, but as efficacious as, butsomewhat less potent than, LPA at LPA1 and LPA2.

NOHPP was also tested for activity at the three cloned LPPs. Althoughthe phosphonate head group eliminates the possibility of NOHPP acting asa substrate, it is possible that the compound could be a competitiveinhibitor. For the initial assay, 0.1, 1.0 and 10.0 mM NOHPP was testedin competition with 50 nM [³²P]LPA and assayed for the appearance ofwater soluble radionuclide. As can be seen in FIG. 3, NOHPP inhibitedLPP3 and, to a lesser extent, LPP1. NOHPP was inactive in this assay atLPP2.

The activity of the alpha keto NOHPP analog (VPC12060) at the three LPPshas also been tested. The structure of the alpha keto NOHPP analog is asfollows:

FIG. 4 presents data concerning the inhibition of the three phosphatases(PAP2a, PAP2b and PAP2c (aka LPP1, LPP3, LPP2, respectively) by thealpha hydroxy phosphonate analog of LPA (VPC12031) and the alpha ketophosphonate analog of LPA (wls060). Both compounds are markedly betterat the PAP2b (LPP3) than PAP2a (LPP1) which in turn is better than PAP2c(LPP2). Based on the results of the activity of NOHPP, it is anticipatedthat analogs of NOHPP will also be active.

EXAMPLE 4 Synthesis of NOHPP [(9Z)-N-(3-hydroxypropyl)Octadec-9-enamide]

To a solution of 1-aminopropanol (4.04 ml, 52.8 mmol) and pyridine (4.27ml, 52.8 mmol) in methylene chloride was slowly added oleoyl chloride (5ml, 15.1 mmol). After 2 hours, the mixture was diluted with chloroformand washed with ammonium chloride (3×), brine (3×) and dried over sodiumsulfate. Column chromatography (15% acetone/chloroform to 50%acetone/chloroform) provided the product as white solid.

(9Z)-N-(3-oxopropyl)Octadec-9-enamide (22)

To a suspension of pyridinium chlorochromate (0.477 g, 2.21 mmol) andsodium acetate (36 mg, 0.44 mmol) in 10 ml of dichloromethane wascannulated as solution of 1 in 8 ml of dichloromethane. The mixture wasstirred for 10 hours, quenched with ether, then filtered through celiteand concentrated to an oil. Chromatography in 15% acetone/chloroformprovided the product in 47% yield (0.495 g).

(9Z)-N-{3-[bis(tert-butoxy)Carbonyl]-3-hydroxypropyl}octadec-9-enamide(23)

To a suspension of sodium hydride in 8 ml of THF at 0° C. was cannula asa solution aldehyde 3 in 6 ml THF. The mixture was allowed equilibrateto room temperature over an hour. The reaction was quenched with waterand acidified with 10% HCl followed by extraction with chloroform (3×).The combined organic extracts were washed with brine (3×) and dried oversodium sulfate. Chromatography in 15% acetone/chloroform provided theproduct as off-white solid (130 mg, 36%).

(9Z)-N-(rac-3-hydroxy-3-phosphonopropyl)Octadec-9-enamide (24)

To a solution of 23 in 1 ml of dichloromethane was added 0.3 mltrifluoroacetic acid. The mixture was stirred for 4 hours (monitored byTLC). Concentration and repeated washings with ether provided theproduct in 100% yield.

EXAMPLE 5

Synthesis of wls-b8L

3-(diethoxycarbonyl)propanenitrile:

In a 50 ml of dry ethanol was added sodium (1.38 g, 60 mmol). Aftercomplete dissolution of sodium, diethyl phosphite (7.75 ml, 60 mmol)dissolved in 20 ml toluene was added slowly. After stirring for 1 hourat room temperature, acrylonitrile (3.95 ml, 57 mmol) dissolved in 20 mltoluene was added dropwise over 1 hour. The mixture was allowed to stirovernight. Dilution with water, extraction with CH₂Cl₂ (3×) and dryingwith sodium sulfate provided a crude yellow oil, which was concentratedand distilled under vacuum (134° C., 1.5 mmHg) to provide the product asclear liquid (6.11 g, 53%).

(3-aminopropyl)diethoxyphosphino-1-one:

To a suspension of 3-(diethoxycarbonyl)propanenitrile (2.78 g, 14.5mmol) and cobalt(II) chloride (0.378 g, 2.91 mmol) in methanol at −30°C. was added sodium borohydride (5.49 g, 145 mmol) in small portions.The mixture was allowed to equilibrate to room temperature and stirredovernight. Concentrated HCl was added until the black mixture turnedblue, which was extracted twice with dichloromethane. The pH of theaqueous phase was adjusted to 9 by addition of concentrated ammoniumhydroxide, and the solution was evaporated to dryness to generatepink/blue solid. Concentrated ammonium hydroxide (100 ml) anddichloromethane (200 ml) was added and stirring was continued for 1hour. Extraction with CH₂Cl₂ (2×), filtration, drying over sodiumsulfate, and concentration provided the product as yellow liquid (2.0 g,70%).

(9Z)-N-[3-(diethoxycarbonyl)propyl]octadec-9-enamide:

To a solution of (3-aminopropyl)diethoxyphosphino-1-one (0.736 g, 3.77mmol) and pyridine (0.61 ml, 7.54 mmol) at 0° C. was slowly addedoleoylchloride (1.25 ml, 3.77 mmol) over 20 minutes. The reactionmixture was allowed to warm to room temperature, followed by stirringfor another 2 hours. Dilution with ethyl acetate and extraction withsaturated aqueous ammonium chloride (3×) and brine (3×), drying oversodium sulfate, and flash chromatography using 15% acetone/chloroformprovided the product as off-white solid (0.572 g, 33%).

(9Z)-N-(3-phosphonopropyl)Octadec-9-enamide:

To a solution of (9Z)-N-[3-(diethoxycarbonyl)propyl]octadec-9-enamide(0.219 g, 0.48 mmol) in acetonitrile was added trimethylsilylbromide(0.189 ml, 1.43 mmol). The reaction mixture was refluxed for 2 hours andthe solvent was evaporated under reduced pressure. Addition of ether andremoval in vacuo, a process repeated several times, provided the productas brown solid (0.192 g, 100%).

EXAMPLE 6

Analysis of wls-b8L Activity:

To assay wls-b8L at individual LPA receptors, the GTPγS binding assaydescribed in Example 2 was utilized. Briefly, individual recombinant LPAreceptors were expressed along with heterotrimeric G proteins in HEK293Tcells. Membranes prepared from these cells are used to measure GTPγ(³⁵S]binding as a function of test compound concentration. The resultingdose-response curves provide relative potency (EC₅₀) and efficacy(E_(max)) for the test compounds. wls-b8L was found to be more potentand efficacious at LPA1 than at either LPA2 or LPA3. Also evident is thelack of receptor-type selectivity by the other phosphonate compounds.

wls-b8L and the other phosphonates were also tested also for activity inthree whole cell assays. For the LPA receptor LPA1, which naturallycouples to Gi/o proteins, rat hepatoma cells (RH7777) were used thathave been transfected only with mouse LPA1 DNA. In these cell cultures,drug dependent inhibition of cAMP accumulation was measured. wls-b8L,was determined to be fully efficacious in this assay at a concentrationof 1 μM. At this concentration wls-b8L is inactive at mobilizing calciumthrough endogenous receptors LPA receptors expressed by MDA MB-231cells. Finally, the activity of wls-b8L in activating a chlorideconductance in Xenopus laevis ooctyes was measured. These cells arenormally activated by LPA through an endogenous receptor. However,wls-b8L does not evoke this response. However, when injected with humanLPA3 or LPA2 mRNAs a very modest response to wls-b8L is detected, butonly when a concentration of 10 μM is applied to the oocyte surface.

EXAMPLE 7

Analysis of VPC12249 Effects in a Renal Ischemia-Reperfusion Model

To examine the effect of the compound VPC12249 on renal injury followingischemia-reperfusion (IR) the following experiment was conducted. C57BL6mice (20 gm, 8 wks of age) were used for all studies. The surgicalprotocol of renal IR has been previously described (Okusa et al., 1999.Am. J. Physiol. (Renal Physiol.) 277: F404-412; Okusa, et al., 2000 Am.J. Physiol. (Renal Physiol.) 279; F809-F818, and Okusa et al., 2001Kidney Int. 59:2114-2125). For the injury protocol, bilateral flankincisions were made under anesthesia with a regimen that consists ofketamine (100 mg/kg, ip.), xylazine (10 mg/kg, ip.) and acepromazine (1mg/kg, im.). Renal pedicles were exposed and cross-clamped for 27-32min. Kidneys were examined for immediate reperfusion following clampremoval. Wounds were closed and mice were returned to cages. Mice wereinjected with VPC12249 (0.01, 0.1 and 1.0 mg/kg/2 hrs) or vehicle. Themice were sacrificed and the kidneys harvested at 1, 4, 8, 24, 48 hrs, 5and 7 days. Treatment with VPC12249 or vehicle was initiated 2 hrs priorto ischemia, at the time of reperfusion and every 2 hrs for 4 additionaldoses. Mice were sacrificed following 24 hrs of reperfusion and plasmawas obtained for BUN and creatinine. Plasma creatinine was 1.49±0.26(n=4), 1.06±0.25 (n=4), 0.45±±0.13 (n=4) and 0.31±0.4 (n=4), forvehicle, VPC12249 0.01, 0.1 and 1.0 mg/kg/2 hrs, respectively. VPC12249administered at 0.1 (P<0.05) and 1.0 mg/kg/2 hrs (P<0.1) weresignificantly different compared to vehicle. H and E sections revealed amarked degree of tissue preservation following VPC12249 at 1 mg/kg/2 hr.

EXAMPLE 8 Synthetic Schemes for Preparing Phospho-Esters Deriviatives

Scheme 1

1. A compound represented by the formula:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, C₈-C₂₂ alkanoyl, C₈-C₂₂ alkenoyl,

wherein m is 0-20; Z is selected from the group consisting of C₃-C₁₀cycloalkyl, C₃-C₁₅ bicycloalkyl, C₅-C₁₀ heterocyclic and aryl; R₁₁ isselected from the group consisting of C₁-C₁₀ alkyl, C₁-C₂₀ alkoxyl,C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino; R₂ and R₃ are independentlyselected from the group consisting of H, C₁-C₆ alkyl, C₂-C₄ alkenyl,C₂-C₄ alkynyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄alkyl)COOR₅, —(C₁-C₁₀ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic,C₇-C₁₂ bicyclic, (C₅-C₈ alkyl)aryl, (C₅-C₈ alkenyl)aryl, (C₅-C₈alkynyl)aryl, and

wherein n is 0-10; R₅ is selected from the group consisting of H andC₁-C₄ alkyl; R₁₂ is selected from the group consisting of halo, C₁-C₁₀alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl,—(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHPR₆ and OR₆; R₁₃ isselected from the group consisting of H, halo, C₁-C₁₀ alkyl, (C₀-C₁₂alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl, —(C₁-C₄alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆; wherein R₆ isselected from the group consisting of C₁-C₁₆ alkyl, C₂-C₁₆ alkenyl,C₂-C₁₆ alkynyl, —(C₁-C₄ alkyl)R₇, —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇and —(C₂-C₄ alkynyl)R₇; and R₇ is selected from the group consisting ofoptionally substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈heterocyclic, optionally substituted C₇-C₁₂ bicyclic and optionallysubstituted C₅-C₈ cycloalkenyl and optionally substituted aryl; y is0-4; and R₄ is represented by the formula

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

 and R₃₀ and R₃, are independently selected from the group consisting ofC₁-C₂ alkoxy,

and pharmaceutically acceptable salts thereof.
 2. The compound of claim1 wherein R¹, is selected from the group consisting of

wherein X is selected from the group consisting of O, CH₂, CHOH, andCHF; R₃₀ and R₃₁ are independently selected from the group consisting of

and y is 0 or
 1. 3. The compound of claim 1 wherein R₁ is selected fromthe group consisting of C₈-C₂₂ alkyl, C₈-C₂₂ alkenyl, C₈-C₂₂ alkanoyl,and C₈-C₂₂ alkenoyl; R₁₃ is H; X is CH₂, CHOH, or CHF and y is
 0. 4. Thecompound of claim 3 wherein n is 1; and R₁₂ is selected from the groupconsisting of SR₆, SOR₆, NHIP₆ and OR₆.
 5. A compound represented by theformula:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl; R₂ is selected from the group consisting of C₁-C₃ alkyl,—(C₂-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —(C₁-C₄ alkyl)COOR₅, —(C₁-C₄alkyl)aryl, and

R₄ is represented by the formula

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

 and R₃₀ and R₃, are independently selected from the group consisting ofC₁-C₂ alkoxy,

R₅ is selected from the group consisting of H and C₁-C₄ alkyl; and R₆ isselected from the group consisting of C₃-C₁₆ alkyl, C₃-C₁₆ alkenyl, and—(C₁-C₄ alkyl)R₇; R₇ is selected from the group consisting of C₃-C₈cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic, C₅-C₈ cycloalkenyl andaryl; and y is 0-4 and pharmaceutically acceptable salts thereof.
 6. Thecompound of claim 5 wherein R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂₁ alkenyl; andR₄ is

wherein X is selected from the group consisting of O, CH₂, CHOH, andCHF; R₃₀ and R₃₁ are independently selected from the group consisting of

and y is 0 or
 1. 7. The compound of claim 6 wherein R₂ is selected fromthe group consisting of C₁-C₃ alkyl, methylene amino, methylene alkyne,phenyl, benzyl, methylene furan, methylene-2-naphthalene, methylenephenol, methylene amino benzyl, methylene phenyl-4-O-benzyl, methylenephenyl-4-benzyl, methylene phenyl-4-chloro, methylenephenyl-4-trans-styrene, methylene phenyl-4-cis-styrene, methylenephenyl-4-O-2,6-dichlorobenzyl and methylene phenyl-4-phenyl.
 8. Thecompound of claim 6 wherein R₄ is

wherein X is selected from the group consisting of O, CH₂, CHOH, andCHF; R₃₀ and R₃₁ are independently selected from the group consisting of

 and R₂ is selected from the group consisting of C₁-C₃ alkyl and benzyl.9. A composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 10. An LPA receptor agonistrepresented by the formula:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl; R₂ is selected from the group consisting of C₁-C₆ alkyl, C₂-C₄alkenyl, C₂-C₄ alkynyl, —(C₂-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —(C₁-C₄alkyl)COOR₅ and aryl; y is 0 or 1; R₄ is represented by the formula

 wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

 and R₃₀ and R₃₁ are independently selected from the group consisting ofC₁-C₂ alkoxy,

 R₅ is selected from the group consisting of H and C₁-C₄ alkyl andpharmaceutically acceptable salts thereof.
 11. The LPA receptor agonistof claim 10 wherein the agonist has the general structure:

wherein R₂ is methyl, ethyl, propyl, isopropyl, butyl, methylene amino,methylene alkyne, phenyl, benzyl, methylene furan,methylene-2-naphthalene; and R₄ is represented by the formula

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

 and R₃₀ and R₃₁ are independently selected from the group consisting ofC₁-C₂ alkoxy,


12. A composition comprising the compound of claim 10 and apharmaceutically acceptable carrier.
 13. An LPA receptor antagonistrepresented by the formula:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; R₂ is selected from the group consisting of H, C₁-C₄alkyl; y is 0 or 1; R₄ is represented by the formula

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

 and R₃₀ and R₃₁ are independently selected from the group consisting ofC₁-C₂ alkoxy,

R₇ is selected from the group consisting of C₃-C₈ cycloalkyl, C₃-C₈heterocyclic, C₇-C₁₂ bicyclic, C₅-C₈ cycloalkenyl and aryl andpharmaceutically acceptable salts thereof.
 14. The compound of claim 13wherein R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂₁ alkenyl; R₂ and R₃ are H; and R₄is

wherein X is selected from the group consisting of O, CH₂, CHOH, andCHF; R₃₀ and R₃₁ are independently selected from the group consisting of


15. The compound of claim 14 wherein the agonist is represented by theformula:


16. The compound of claim 13 wherein R₂ and R₃ are independentlyselected from the group consisting of H, benzyl, methylene furan,methylene-2-naphthalene, methylene phenyl-4-O-benzyl, methylenephenyl-4-benzyl, methylene phenyl-4-chloro, methylenephenyl-4-trans-styrene, methylene phenyl-4-cis-styrene, methylenephenyl-4-O-2,6-dichlorobenzyl and methylene phenyl-4-phenyl.
 17. Alyso-lipid phosphate phosphatase resistant LPA analog, said agonistrepresented by the formula

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, C₈-C₂₂ alkanoyl, C₈-C₂₂ alkenoyl,

wherein m is 0-20; Z is selected from the group consisting of C₃-C₁₀cycloalkyl, C₃-C₁₅ bicycloalkyl, C₅-C₁₀ heterocyclic and aryl; R₁₁ isselected from the group consisting of C₁-C₁₀ alkyl, C₁-C₂₀ alkoxyl,C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino; R₂ and R₃ are independentlyselected from the group consisting of H, hydroxy, C₁-C₆ alkyl, C₂-C₄alkenyl, C₂-C₄ alkyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅,—(C₁-C₄ alkyl)COOR₅, —(C₁-C₄ alkyl)aryl, C₃-C₈ cycloalkyl, C₃-C₈heterocyclic, C₇-C₁₂ bicyclic, (C₅-C₁₀ alkyl)aryl, (C₅-C₈ alkenyl)aryl,(C₅-C₈ alkynyl)aryl, and

wherein n is 0-10; R₅ is selected from the group consisting of H andC₁-C₄ alkyl; R₁₂ is selected from the group consisting of halo, C₁-C₁₀alkyl, (C₀-C₁₂ alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl,—(C₁-C₄ alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆; R₁₃ isselected from the group consisting of H, halo, C₁-C₁₀ alkyl, (C₀-C₁₂alkyl)aryl, (C₂-C₁₂ alkenyl)aryl, (C₂-C₁₂ alkynyl)aryl, —(C₁-C₄alkyl)OH, —(C₂-C₁₂ alkenyl)OH, SR₆, SOR₆, NHR₆ and OR₆; wherein R₆ isselected from the group consisting of C₁-C₁₆ alkyl, C₂-C₁₆ alkenyl,C₂-C₁₆ alkynyl, —(C₁-C₄ alkyl)R₇, —(C₂-C₄ alkenyl)R₇, —(C₁-C₄ carboxy)R₇and —(C₂-C₄ alkynyl)R₇; and R₇ is selected from the group consisting ofoptionally substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈heterocyclic, optionally substituted C₇-C₁₂ bicyclic, optionallysubstituted C₅-C₈ cycloalkenyl and optionally substituted aryl; q is0-4; R₈ and R₉ are independently selected from H, hydroxyl, amino, COOH,halo, —PO₃; or R₈ and R₉ taken together form a keto group or a methylenegroup; R₁₀ is selected from the group consisting of O, S and NH; and R₃₀and R₃₁ are independently selected from the group consisting of C₁-C₂alkoxy,

and pharmaceutically acceptable salts thereof.
 18. The compound of claim17 wherein R₁ is selected from the group consisting of

R₁₃ and R₁₀ are H; and q is
 1. 19. The compound of claim 17 wherein R₁is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂ alkenyl,C₈-C₂₂ alkanoyl, and C₈-C₂₂ alkenoyl; R₁₃ and R₁₀ are H; and q is
 1. 20.The compound of claim 19 wherein n is 1; and R₁₂ is selected from thegroup consisting of SR₆, SOR₆, NHR₆ and OR₆
 21. A lyso-lipid phosphatephosphatase resistant LPA analog, said agonist represented by theformula

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; R₂ and R₃ are independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, —(C₁-C₄ alkyl)COOR₅, and

R₅ is selected from the group consisting of H and C₁-C₄ alkyl; and R₆ isselected from the group consisting of C₃-C₁₆ alkyl, C₃-C₁₆ alkenyl,—(C₁-C₄ alkyl)R₇; R₇ is selected from the group consisting of C₃-C₈cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic and C₅-C₈ aryl; R₈ andR₉ are independently selected from the group consisting of H, C₁-C₄alkyl, and —PO₃, or R₈ and R₉ are combined to form a keto group; and R₃₀and R₃₁ are independently selected from the group consisting of C₁-C₂alkoxy,

and pharmaceutically acceptable salts thereof.
 22. The analog of claim21 wherein R₁ is C₁₃-C₁₇ alkyl or C₁₇-C₂₁ alkenyl; and R₃, R₈ and R₉ areH.
 23. A lyso-lipid phosphate phosphatase resistant LPA receptor agonistrepresented by the formula:

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; R₂ is selected from the group consisting of H, hydroxyl,C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄alkyl)NH₂, —COOR₅, —(C₁-C₄ alkyl)COOR₅, and benzyl; and R₉ is selectedfrom the group consisting of H, C₁-C₄ alkyl, halo and —PO₃; and R₃₀ andR₃₁ are independently selected from the group consisting of C₁-C₂alkoxy,

and pharmaceutically acceptable salts thereof.
 24. A compositioncomprising the compound of claim 17 and a phamaceutically acceptablecarrier.
 25. A method of enhancing wound repair, said method comprisingthe steps of contacting the wound with a composition comprising acompound represented by the formula:

 wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; R₂ is selected from the group consisting of C₁-C₆ alkyl,C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₂-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂,—(C₁-C₄ alkyl)COOR₅ and benzyl; y is 0 or 1; R₄ is represented by theformula

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

 and R₃₀ and R₃₁ are independently selected from the group consisting ofC₁-C₂ alkoxy,

and R₅ is selected from the group consisting of H and C₁-C₄ alkyl. 26.The method of claim 25 wherein R₂ is methyl, ethyl, propyl, isopropyl,butyl, methylene amino, methylene alkyne, phenyl, benzyl, methylenefuran, methylene-2-naphthalene; and R₄ is represented by the formula

wherein X is selected from the group consisting of O, CH₂, CHOH and CHF;and R₃₀ and R₃₁ are independently selected from the group consisting ofC₁-C₂ alkoxy,


27. A method of treating a disease characterized by cell hyperproliferation, said method comprising the step of administering to apatient a composition comprising a compound represented by the formula

wherein R₁ is selected from the group consisting Of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; y is 0 or 1; R₄ is represented by the formula

wherein R₁₂ is selected from the group consisting of O and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

R₃₀ and R₃, are independently selected from the group consisting ofC₁-C₂ alkoxy,

R₆ is selected from the group consisting of C₃-C₁₆ alkyl, C₃-C₁₆alkenyl, —(C₁-C₄ alkyl)R₇; and R₇ is selected from the group consistingof C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic, C₅-C₈cycloalkenyl and aryl, with the proviso that R₂ and R₃ are not both H.28. A method of treating reperfusion type injury, said method comprisingthe step of administering to a patient a composition comprising acompound represented by the formula

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; y is 0 or 1; R₄ is represented by the formula

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH, S, CH₂, CHOH, CO₂H, CHF,CF₂, and

R₃₀ and R₃₁ are independently selected from the group consisting ofC₁-C₂ alkoxy,

R₆ is selected from the group consisting of C₃-C₁₆ alkyl, C₃-C₁₆alkenyl, —(C₁-C₄ alkyl)R₇; and R₇ is selected from the group consistingof C₃-C₈ cycloalkyl, C₃-C₈ heterocyclic, C₇-C₁₂ bicyclic, C₅-C₈cycloalkenyl and aryl, with the proviso that R₂ and R₃ are not both H.29. The method of claim 28 wherein the composition is administered priorto injury to prevent tissue damage.
 30. The method of claim 28 whereinthe composition is administered after injury to prevent or limit tissuedamage.
 31. A method of inhibiting LPP activity, said method comprisingthe step of administering a compound represented by the formula

wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl andC₈-C₂₂ alkenyl; R₂ and R₃ are independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₂-C₄ alkenyl, C₂-C₄ alkynyl, —(C₁-C₄alkyl)OH, —(C₁-C₄ alkyl)NH₂, —COOR₅, and —(C₁-C₄ alkyl)COOR₅; R₅ isselected from the group consisting of H and C₁-C₄ alkyl; and R₈ and R₉are independently selected from the group consisting of H, halo, C₁-C₄alkyl, and —PO₃, or R₈ and R₉ are combined to form a keto group and R₃₀and R₃₁ are independently selected from the group consisting of C₁-C₂alkoxy,


32. The method of claim 31 wherein R₂ is H